Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors

Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.     

Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia

Internal ribosome entry site (IRES)-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), human rhinovirus (HRV) or poliovirus (PV). Under mild hypothermic conditions (30 and 35°C), we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB) and upstream of N-Ras (unr), the IRES trans-acting factors (ITAFs) of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.     

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.     

The efficiency of different IRESs (Internal Ribosomes Entry Site) in monocistronic mRNAS

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.     

Replication of poliovirus RNA with complete internal ribosome entry site deletions

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.     

La autoantigen enhances translation of BiP mRNA

Translational initiation of the human BiP mRNA is directed by an internal ribosomal entry site (IRES) located in the 5′-untranslated region (5′-UTR). In order to understand the mechanism of the IRES-dependent translation of BiP mRNA, cellular proteins interacting with the BiP IRES were investigated. La autoantigen, which augments the translation of polioviral mRNA and hepatitis C viral mRNA, bound specifically to the second half of the 5′-UTR of the BiP IRES and enhanced translation of BiP mRNA in both in vitro and in vivo assays. This finding suggests that cellular and viral IRESs containing very different RNA sequences may share a common mechanism of translation.     

Giardiavirus Internal Ribosome Entry Site Has an Apparently Unique Mechanism of Initiating Translation

Giardiavirus (GLV) utilizes an internal ribosome entry site (IRES) for translation initiation in the early branching eukaryote Giardia lamblia. Unlike most of the viral IRESs among higher eukaryotes, which localize primarily within the 5′-untranslated region (UTR), the GLV IRES comprises 253 nts of 5′UTR and the initial 264 nts in the open-reading-frame (ORF). To test if GLV IRES also functions in higher eukaryotic systems, we examined it in rabbit reticulocyte lysate (RRL) and found that it functions much less efficiently than the IRES from the Encephalomyocarditis virus (EMCV) or Cricket paralysis virus (CrPV). In contrast, both EMCV-IRES and CrPV-IRESs were inactive in transfected Giardia cells. Structure-function analysis indicated that only the stem-loop U5 from the 5′UTR and the stem-loop I plus the downstream box (Dbox) from the ORF of GLV IRES are required for limited IRES function in RRL. Edeine, a translation initiation inhibitor, did not significantly affect the function of GLV IRES in either RRL or Giardia, indicating that a pre-initiation complex is not required for GLV IRES–mediated translation initiation. However, the small ribosomal subunit purified from Giardia did not bind to GLV IRES, indicating that additional protein factors may be necessary. A member of the helicase family IBP1 and two known viral IRES binding proteins La autoantigen and SRp20 have been identified in Giardia that bind to GLV IRES in vitro. These three proteins could be involved in facilitating small ribosome recruitment for initiating translation.     

Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment

The 5′ UTR of c-myc mRNA contains an internal ribosome entry segment (IRES) and consequently, c-myc mRNAs can be translated by the alternative mechanism of internal ribosome entry. However, there is also some evidence suggesting that c-myc mRNA translation can occur via the conventional cap-dependent scanning mechanism. Using both bicistronic and monocistronic mRNAs containing the c-myc 5′ UTR, we demonstrate that both mechanisms can contribute to c-myc protein synthesis. A wide range of cell types are capable of initiating translation of c-myc by internal ribosome entry, albeit with different efficiencies. Moreover, our data suggest that the spectrum of efficiencies observed in these cell types is likely to be due to variation in the cellular concentration of non-canonical translation factors. Interestingly, the c-myc IRES is 7-fold more active than the human rhinovirus 2 (HRV2) IRES and 5-fold more active than the encephalomyocarditis virus (EMCV) IRES. However, the protein requirements for the c-myc IRES must differ significantly from these viral IRESs, since an unidentified nuclear event appears to be a pre-requisite for efficient c-myc IRES-driven initiation.     

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.     

A Phylogenetically Conserved Stem-Loop Structure at the 5′ Border of the Internal Ribosome Entry Site of Hepatitis C Virus Is Required for Cap-Independent Viral Translation

Hepatitis C virus (HCV) initiates translation of its polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of the 341-nucleotide (nt) 5′ nontranslated RNA (5′NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which secondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5′ border of the HCV IRES and thus may function in viral translation. This prediction was tested by a mutational analysis of putative helical structures that examined the impact of both covariant and noncovariant nucleotide substitutions on IRES activity in vivo and in vitro. Results of these experiments provide support for predicted base pair interactions between nt 44 to 52 and 111 to 118 and between nt 65 to 70 and 97 to 102 of the HCV 5′NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted in reciprocal changes in V1 nuclease cleavage patterns within the opposing strand of the putative helix, consistent with the predicted base pair interactions. IRES activity was highly dependent on maintenance of the stem-loop II structure but relatively tolerant of covariant nucleotide substitutions within predicted helical segments. Sequence alignments suggested that the deduced domain II structure is conserved within the IRESs of pestiviruses as well as the novel flavivirus GB virus B. Despite marked differences in primary nucleotide sequence within conserved helical segments, the sequences of the intervening single-stranded loop segments are highly conserved in these different viruses. This suggests that these segments of the viral RNA may interact with elements of the host translational machinery that are broadly conserved among different mammalian species.     

La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro

Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.     

IRES-driven translation is stimulated separately by the FMDV 3'-NCR and poly(A) sequences

The 3′ end region of foot-and-mouth disease virus (FMDV) consists of two distinct elements, a 90 nt untranslated region (3′-NCR) and a poly(A) tract. Removal of either the poly(A) tract or both the 3′-NCR and the poly(A) tract abrogated infectivity in susceptible cells in the context of a full-length cDNA clone. We have addressed the question of whether the impairment of RNA infectivity is related to defects at the translation level using a double approach. First, compared to the full-length viral RNA, removal of the 3′ sequences reduced the efficiency of translation in vitro. Secondly, a stimulatory effect of the 3′ end sequences on IRES-dependent translation was found in vivo using bicistronic constructs. RNAs carrying the FMDV 3′ end sequences linked to the second cistron showed a significant stimulation of IRES-dependent translation, whereas cap-dependent translation was not affected. Remarkably, IRES-dependent stimulation exerted by the poly(A) tract or the 3′-NCR seems to be the result of two separate events, as the 3′-NCR alone enhanced IRES activity on its own. Under conditions of FMDV Lb protease-induced translation shut-off, the stimulation of IRES activity reached values above 6-fold in living cells. A northern blot analysis indicated that IRES stimulation was not the consequence of a change in the stability of the bicistronic RNA produced in transfected cells. Analysis of the RNA-binding proteins interacting with a mixture of 3′ end and IRES probes showed an additive pattern. Altogether, our results strongly suggest that individual signals in the viral 3′ end ensure stimulation of FMDV translation.     

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3 D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3 D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1(DDX1), which interacted with FMDV 3 D. The DDX1-3 D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA) in porcine kidney 15(PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-b activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.     

Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

A virus‐derived microRNA targets immune response genes during SARS‐CoV‐2 infection

SARS‐CoV‐2 infection results in impaired interferon response in patients with severe COVID‐19. However, how SARS‐CoV‐2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS‐CoV‐2‐infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus‐derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3′UTR of interferon‐stimulated genes and represses their expression in a miRNA‐like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID‐19 patients. We propose that SARS‐CoV‐2 can potentially employ a virus‐derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon‐mediated immune response.     

RNA‐binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral activity

The type I interferon (IFN‐I, IFN‐α/β)‐mediated immune response is the first line of host defense against invading viruses. IFN‐α/β binds to IFN‐α/β receptors (IFNARs) and triggers the expression of IFN‐stimulated genes (ISGs). Thus, stabilization of IFNARs is important for prolonging antiviral activity. Here, we report the induction of an RNA‐binding motif‐containing protein, RBM47, upon viral infection or interferon stimulation. Using multiple virus infection models, we demonstrate that RBM47 has broad‐spectrum antiviral activity in vitro and in vivo. RBM47 has no noticeable impact on IFN production, but significantly activates the IFN‐stimulated response element (ISRE) and enhances the expression of interferon‐stimulated genes (ISGs). Mechanistically, RBM47 binds to the 3''UTR of IFNAR1 mRNA, increases mRNA stability, and retards the degradation of IFNAR1. In summary, this study suggests that RBM47 is an interferon‐inducible RNA‐binding protein that plays an essential role in enhancing host IFN downstream signaling.     

La autoantigen suppresses IRES-dependent translation of the hepatitis A virus

The human RNA-binding protein La, is an essential trans-acting factor in IRES-dependent translation initiation of poliovirus, the prototypic picornavirus. For hepatitis A virus (HAV), an unusual member of this virus family, the role of host proteins in its inefficient translation and slow replication is unclear. Using small interfering RNA in vivo and purified La in vitro, we demonstrate for the first time that La suppresses HAV IRES-mediated translation and replication. We show that La binds specifically to distinct parts of the HAV IRES and that—unlike poliovirus—HAV proteinase 3C does not cleave La. The La-mediated suppression of HAV translation and stimulation of poliovirus translation implies unexpected mechanistic differences between viral IRES elements.     

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A^pro or L^pro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A^pro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L^pro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A^pro and L^pro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L^pro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.